Proc. Natl. Acad. Sci. USAVol. 85, pp. 5966-5970, August 1988Cell Biology

Cleavage furrow isolated from newt eggs: Contraction, organizationof the actin filaments, and protein components of the furrow

(cell division/contractile arc/microfilaments/fMlament bundles/plasma membrane)

ISSEI MABUCHI*, SHOICHIRo TSUKITAt, SACHIKO TSUKITAt, AND TSUYOSHI SAWAIt*Department of Biology, College of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153; tDepartment of Ultrastructural Research, TheTokyo Metropolitan Institute for Medical Sciences, Honkomagome, Bunkyo-ku, Tokyo 113; and tDepartment of Biology, Faculty of General Education,Yamagata University, Yamagata 990, Japan

Communicated by Daniel Mazia, May 2, 1988

ABSTRACT The cleavage-furrow region was isolated sur-gically from newt eggs at the early stage of the first cleavage.The isolated furrow contracted in the presence of ATP at aCa2l concentration of 10 or 100 nM, although the speed wasless than that of the furrow in vivo. Cytochalasin B, cytocha-lasin D, phalloidin, p-chloromercuribenzoate, and N-ethyl-maleimide interfered with the contraction, but colchicine didnot. The furrow contained bundles of actin filaments ofopposite polarities oriented parallel to the long axis of thefurrow; these bundles may be the main component of thecontractile arc. From electron microscopic observation of thinsections of the furrow, it was suggested that the actin bundlesof the contractile arc were organized from preexisting corticalrfaments that were connected to the plasma membrane bygranular materials at their barbed ends. Contractile-arc actinlaments were revealed to be crosslinked by thin strands by the

rapidfreezing/deep etching-replication technique. Two-dimen-sional polyacrylamide gel electrophoresis showed that severalproteins found in the furrow cortex are absent from the corticallayer before the cleavage furrow is formed.

The mechanism ofcytokinesis in animal cells has been a greatproblem in cell biology. Recent efforts using electron mi-croscopy, immunofluorescence microscopy, and microinjec-tion techniques lead us to conclude that the motive force ofcleavage is generated by the interaction between actinfilaments and myosin in the contractile ring (symmetriccleavage) or contractile arc (asymmetric cleavage) in thecleavage furrow (1, 2). However, the process of formation ofthe contractile ring (arc), its ultrastructure, its spatial rela-tionship with the plasma membrane, and the mode of itscontraction are still mysterious. Glycerol- or detergent-extracted cleavage models have been developed (3-5), butthey are insufficient to solve these problems. Isolation andcharacterization of the cleavage furrows from dividing cellswould be an ideal approach to solving these problems, butisolation ofthe furrow was thought to be difficult because thisapparatus is a transitory, and therefore a quite labile, struc-ture. However, Perry et al. (6) excised cleavage-furrowcortex from a dividing newt egg and showed that thepreparation contained microfilaments. Here we demonstratethat cleavage furrows isolated surgically from newt eggscontract upon addition of ATP, possess bundles of cross-linked actin filaments that are formed from preexistingmembrane-linked actin filaments, and contain proteins thateither are not detected or are detected in lesser amounts in thebulk cortex.

MATERIALS AND METHODSIsolation of Cleavage Furrow. Shedding of fertilized eggs of

the newt Cynops pyrrhogaster was induced by injection ofgonadotropic hormone (Gonatropin, Teikoku Zoki, Tokyo).Eggs were freed from the jelly envelope by using smallscissors or by brief treatment with 88 mM sodium thio-glycolate at pH 10 and then were placed in cleavage-furrowisolation medium [0.25 M sucrose/0.1 M KCI/4 mMMgSO4/1.1 mM EGTA/0. 1 mM CaCl2/10mM 4-morpholine-propanesulfonate (Mops) buffer, pH 7.2]. When the firstcleavage started, eggs were transferred to a shallow depres-sion in an agar gel that coated the bottom of a glass dish filledwith the isolation medium. When the length of the furrowreached 0.1-0.3 mm, the vitelline coat was removed with twopairs of tweezers under a dissection microscope. Incisionswere made with a fine glass needle on either side of thepigmented thread at the bottom of the furrow cortex (6). Thenthe furrow was dissected from the egg by cutting both endswith two fine glass needles. These crosscuts were made 50-70 ,um from the edges of the furrow. Care was taken not topull the furrow during isolation.

Electron Microscopy. Negative staining was performedwith 1% (wt/vol) uranyl acetate. For thin sectioning, speci-mens were fixed with 0.5% (wt/vol) tannic acid/2.5% (wt/vol) glutaraldehyde/0.1 M cacodylate buffer, pH 7.5, post-fixed with 1% (wt/vol) OSO4, and embedded in Epon 812(Polyscience, Warrington, PA). For etching-replication, thefurrow was rapidly frozen with liquid helium, fractured, anddeeply etched as described (7). Specimens were examined at100 kV with a 100CX or 1200EX electron microscope (JEOL,Tokyo).

Two-Dimensional PAGE. Isolated cleavage furrows weretransferred immediately after isolation into 90% (vol/vol)acetone at 0C. After 60 furrows were collected, they werepelleted by centrifugation at 500 x g for 2 min, washed twicewith 90% acetone and twice with 100% acetone, and dried.For comparison, the bulk cortical layer was isolated, stored,and processed similarly.Two-dimensional PAGE was carried out according to

O'Farrell (8) with modifications. The first-dimension gel,containing 2% (wt/vol) Ampholines (LKB, pH 5-7/pH 3.5-10 weight ratio, 4:1), was made in a hematocrit tube (cut at3.5 cm long) at a height of 3 cm. The gel was solidified withoutO'Farrell's gel-overlay solution.Dried cleavage furrows were dissolved in 3 gl of O'Far-

rell's lysis buffer by sonication. After application of thesample onto the first-dimension gel, the upper electrodesolution (10 mM NaOH) was directly overlaid. Isoelectricfocusing was carried out at 250 V for 2 hr. Two gel rods, one

Abbreviation: NBD-phallacidin, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin.

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containing cleavage-furrow proteins and the other containingcortical proteins, were placed in tandem on the same gel slab(10% acrylamide; 0.8 mm thick, 7 cm wide, and 5 cm inheight), which contained 0.1% NaDodSO4. After electropho-resis, the gel slab was stained with ammoniacal silver (9).Marker proteins used were rabbit skeletal muscle actin andtropomyosin, chicken breast muscle a-actinin, and sea urchinsperm flagellar tubulin.

RESULTSFurrows were isolated at the early cleavage stage by asurgical operation (6) from eggs of the newt C. pyrrhogaster.An isolation buffer containing no ATP was chosen so that theionic conditions and the osmotic pressure were similar tothose in the cytoplasm. The free Ca2+ concentration wasregulated to be 10 nM (pCa = 8). Under these isolationconditions, light microscopy showed that the morphology ofthe furrow did not change significantly after isolation (Fig.la). The contractility of the isolated furrow was investigatedas follows. Two points were arbitrarily determined on thefurrow. The change in the distance between these points was

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FIG. 1. Contraction of isolated furrow. (a) Just after isolation,furrow was transferred to a well of a glass slide containing isolationmedium and was examined with a phase-contrast microscope.Numbers are time after transfer (min). (b) Same as a except that theisolation medium had been supplemented with 2 mM ATP. (x 160;bar = 0.2 mm.) Graphs are plots of the distance between twoarbitrary points on the furrow (arrowheads) versus time aftertransfer.

monitored. In the absence of ATP, only a slight contractionwas observed (Fig. la; Table 1). However, when the furrowwas transferred to a medium containing ATP, it contracted,especially at portions near the ends. The initial rate ofcontraction in the example shown in Fig. lb was determinedto be 10 tum/min. This was about one-third of the contractionrate of the furrow in situ (T.S., unpublished data). Theaverage rate obtained from all parts of the isolated furrowfrom similar determinations was 3.4 Am/min (Table 1).Increases in the Ca2+ concentration up to 0.1 ,uM did notseem to affect the rate, but the rate was diminished at 1 ,MCa2 + (Table 1). We could not check the effect of higher Ca2 +concentrations on the ATP-dependent contraction becausethe furrow contracted without ATP and crumpled within 30sec after isolation when the Ca2+ concentration was >10AM. The rate was also diminished in the absence (

5968 Cell Biology: Mabuchi et al.

Table 1. Contraction of isolated cleavage furrowContraction, ,tm/min [mean ± SD (n)]

Inhibitor pCa + ATP - ATPNone >9* -1.59 ± 1.13 (13) -0.75 ± 0.74 (10)

8 -3.42 ± 2.55 (40) -0.29 ± 1.66 (27)7 -3.25 ± 1.85 (5) 0.75 ± 0.77 (4)6 -1.82 ± 0.98 (7) 0.65 ± 2.84 (6)

Colchicine (0.1 mM) 8 -5.72 ± 5.43 (10) -0.47 ± 1.14 (8)Cytochalasin D (20 ,ug/ml) 8 -0.64 ± 1.09 (17) 0.50 ± 0.59 (4)Cytochalasin B (10 ,ug/ml) 8 -0.28 ± 0.28 (5)Phalloidin (50 ,uM) 8 0.19 ± 1.40 (18)p-Chloromercuribenzoate (50 ,uM) 8 -0.02 ± 0.33 (6) 0.50 (2)N-Ethylmaleimide (0.25 mM) 8 -0.19 ± 0.21 (4)

Cortical stripst 8 1.38 ± 2.26 (13) -0.77 ± 1.64 (12)The rate of change in the distance between two arbitrarily determined points of the isolated furrows

was recorded. A negative value indicates that the distance became smaller; a positive value indicatesthat the distance became larger. Figures in parentheses are numbers of determinations.

2+*Isolation medium contained 2 mM EGTA and no added CatCortical strips that had originally been located 0.2-0.3 mm away from and parallel to the furrow. Thesestrips were tested in the absence of inhibitor.

sition from unorganized filaments into a bundle was observed.In a longitudinal view of a replica of the contractile arc, theparallel actin filaments were seen to be crosslinked by thinstrands. At least two kinds of strands were recognized, oneabout 15 nm long and the other 30-40 nm long (Fig. 3g).

Isolated furrows were analyzed by two-dimensional PAGEon the same gel slab with cortices of the animal hemispherethat had been isolated at a stage before the formation of thecleavage furrow (Fig. 4). On the basis of the mobility ofmarker proteins, actin, tubulin, a-actinin and tropomyosinswere tentatively identified. There seemed to be no significantdifference between the cortex and the cleavage furrow in thecontent of actin, a-actinin, and tropomyosins. Several pro-

teins were unique to the furrow (Mr 98,000 protein, tubulin,Mr 42,000-44,000 proteins) or were more abundant in thefurrow than in the cortex (Mr 67,000 protein). The Mr 120,000protein seemed to become acidic in the furrow. There werethree Mr 56,000 proteins that were not detected in the cortex.Two of them (56-1 and 56-2 in Fig. 4) may be basic forms ofthe Mr 56,000 protein in the cortex, whereas 56-3 may be anacidic form.

DISCUSSIONThe following results were obtained by light and electronmicroscopic observations of isolated cleavage furrows fromnewt eggs. (i) Its contraction required ATP. This confirms the

FIG. 2. Actin bundles in isolated cleavage furrow. (a) Triton X-100-washed furrow observed with Nomarski differential interference optics.Furrow was placed in isolation medium containing 0.5% Triton X-100 and 0.3 ,uM N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBDphallacidin; Molecular Probes, Eugene, OR). (b) NBD-phallacidin staining of the isolated furrow. The same furrow shown in a was examinedunder epifluorescence optics. ( x 400; bar = 0.1 mm.) (c) Negatively stained images of the isolated furrow. The isolated cleavage furrow wasfrayed at the air/water interface, immediately mounted on a carbon-coated Formvar grid, and stained negatively with 1%6 aqueous uranyl acetate.(d) Binding ofmyosin subfragment 1 to filaments in the isolated furrow. The furrow on the grid was incubated for 1 min with myosin subfragment1 at 2 mg/ml in isolation medium, washed with drops of the isolation medium, and stained negatively with 1% uranyl acetate. Directionalityof the arrowhead structure is indicated (arrows). ( x 49,500; bar = 1 ,um.)

Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 3. Ultrastructure of cleavage furrow. (a) Overall cross-sectional view of an isolated furrow. Arrows indicate the edges of themicrofilamentous layer. E, extracellular side. I, intracellular side. ( x 6600; bar = 5 ,.m.) (b) Longitudinal view of an isolated furrow. Arrowindicates plasma membrane. (x40,000.) (c) Oblique view of an isolated furrow. (x40,000.) (d) A region apart from the edge of themicrofilamentous layer in an isolated furrow. Actin filaments are not organized. Arrows indicate the attachment site of filaments, which appearsas a granular mass. (x 155,000.) (e) A region similar to d in a furrow incubated with myosin subfragment 1. Arrows indicate the directionalityof the "arrowhead" structures. ( x 155,000.) (f) Longitudinal view of an edge of the microfilamentous layer. Actin bundles are being organizedfrom top to bottom. B, actin bundle. (x 60,000.) (g) Deep etch-replica image of the isolated furrow. This picture is a longitudinal view of thecontractile-arc region. Arrowheads indicate long crosslinkers, and arrows indicate short ones. Note the 5.5-nm genetic helix of actin filaments.( x 180,000.) (Bars in b-f = 0.1 ,gm.)

observation by Hoffman-Berling (3) that the glycerinateddividing cultured fibroblast advanced its furrow upon addi-

tion ofATP. However, in Hoffman-Berling's experiment, theCa2+ concentration was not regulated. Studies using deter-

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Cell Biology: Mabuchi et al.

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5970 Cell Biology: Mabuchi et al.

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FIG. 4. Two-dimensional PAGE of isolated furrows. (a) Fur-rows. (b) Cortical layer of the egg from the animal hemisphere beforethe formation of the cleavage furrow. Polarity of isoelectric focusing(first dimension) is indicated (- and +). Marker proteins (lane atleft) used for the second dimension were phosphorylase a, bovineserum albumin, rabbit skeletal muscle actin, carbonic anhydrase, andsoybean trypsin inhibitor. Numbers are M, x 10o. A, actin. T,tubulin. TM, tropomyosins. a, a-actinin-like spot.

gent-extracted division models suggested that micromolarCa2 + interferes with cleavage (4, 5), and 10 nM was reportedto be the best Ca2 + concentration for sea urchin eggs (5). Ourobservation that the rate of contraction of the isolated furrowwas faster at 10 or 100 nM Ca2" than at 1 AM or